Recombinant expression of methioninase and uses in managing cancer

Abstract

Disclosed herein are nucleic acids and vectors encoding methioninase for uses in treating cancer. In certain embodiments, expression of methioninase is controlled using loxP sites and Cre enzymes allowing for cancer cell type targeting. In certain embodiments, this disclosure relates to method of treating cancer comprising administering an effective amount of a recombinant viral vector encoding methioninase in combination with administration of a Cre enzyme or nucleic acid or vector encoding the same to a subject in need thereof. In certain embodiments, this disclosure relates to pharmaceutical compositions comprising nucleic acids and vectors encoding methioninase and / or a Cre enzyme as reported herein.

Description

[0001] RECOMBINANT EXPRESSION OE METHIONINASE AND USES IN MANAGING CANCER

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 771,998 filed March 14, 2025. The entirety of this application is hereby incorporated by reference for all purposes.

[0004] INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN XML FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM

[0005] The Sequence Listing associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 25005PCT.xml. The XML file is 19,843 bytes, was created on March 12, 2026, and is being submitted electronically via the USPTO Patent Center.

[0006] BACKGROUND

[0007] Medulloblastoma (MB) is a common type of primary brain cancer in children. It originates in the lower back part of the brain called the cerebellum, the part of the brain responsible for balance and coordination. MB can happen at any age, but most often occurs in young children. The 5-year survival rate is approximately 80%, though survival rates can vary greatly depending on age, spread, recurrence, and subtype. Thus, there is a need to identify improved therapies.

[0008] In humans, methionine is used as a key component of all proteins, and methionine is also metabolized by enzymatic conversion to S-adenosyl-methionine (SAM). SAM is utilized in numerous metabolic reactions. In contrast to humans, bacteria produce methionine gamma lyase (also referred to as “methioninase” or “METase”) an enzyme that degrades methionine to a-ketobutyrate, methanethiol, and ammonia. See e.g., Pasut et al. Adv Drug Delv Rev, 60 (2008) 69-78.

[0009] Han et al. report oral dosing of recombinant methioninase in patients with bone-metastatic prostate cancer and ovarian cancer. Anti-Cancer Res, 2020, 40: 2813-2819.

[0010] Javia et al. provide a review on L-methioninase in cancer therapy. Int J Biol Macro, 2024, 265, 130997.Kawaguchi et al. report oral recombinant methioninase (o-rMETase) is superior to injectable (rMETase) and overcomes acquired gemcitabine resistance in pancreatic cancer. Cancer Let, 2018, 432, 251-259.

[0011] Several clinical trials using methionine-restricted diets were terminated due to the difficulty of maintaining such diets. See e.g., Mattes et al. A Phase I Trial of a Methionine Restricted Diet with Concurrent Radiation Therapy, Nutrition and Cancer, 2024, 76:6, 463-468.

[0012] Kubota et al. report recombinant-methioninase-producing Escherichia coli inhibits triplenegative breast cancer. Cancer Dia & Prog, 2023, 3:649-654.

[0013] See also US Pat. Nos. 9,279,119, 11,001,826, and 11,371,036.

[0014] SUMMARY

[0015] Disclosed herein are nucleic acids and vectors encoding methioninase for uses in treating cancer. In certain embodiments, expression of methioninase is controlled using loxP sites and Cre enzyme allowing for cancer cell type targeting. In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of a recombinant vector encoding methioninase in combination with administration of a Cre enzyme or nucleic acid or vector encoding the same to a subject in need thereof. In certain embodiments, this disclosure relates to pharmaceutical compositions comprising nucleic acids and vectors encoding methioninase and / or a Cre enzyme.

[0016] In certain embodiments, this disclosure relates to a recombinant viral vector comprising a segment encoding methioninase and two pairs of loxP sites. In certain embodiments, the loxP sites have the nucleotide sequence of

[0017] 5’-ATAACTTCGTATANNNTANNNTATACGAAGTTAT (SEQ ID NO: 1) wherein N is any nucleotide.

[0018] In certain embodiments, a first pair of loxP sites have the nucleic acid sequence of 5’-ATAACTTCGTATAGCATACATTATACGAAGTTATACTAGTGCTATCGATGTTG AGCTCAGGTACCTATAACTTCGTATAGGATACTTTATACGAAGTTAT (SEQ ID NO: 2) In certain embodiments, a second pair of loxP sites have the nucleic acid sequence of 5’- ATAACTTCGTATAATGTATGCTATACGAAGTTATCAAGCTTGCGTACGCGTGA CCGGTACTCGAGGATAACTTCGTATAAAGTATCCTATACGAAGTTAT (SEQ ID NO: 3).In certain embodiments, the viral vector is a recombinant Murine Stem Cell Virus (MSCV) vector.

[0019] In certain embodiments, this disclosure relates to a recombinant vector disclosed herein wherein the segment encoding methioninase comprises an N-terminal segment encoding a secretion sequence. In certain embodiments, the secretion sequence is an IL2 secretion sequence having the amino acid sequence of MYSMQLASCVTLTLVLLVNS (SEQ ID NO: 5) or variant thereof.

[0020] In certain embodiments, this disclosure relates to a recombinant vector disclosed herein wherein the encoded methioninase has the amino acid sequence of MRDSHNNTGFSTRAIHHGYDPLSHGGALVPPVYQTATYAFPTVEYGAACFAGEE AGHFYSRISNPTLALLEQRMASLEGGEAGLALASGMGAITSTIWTLLRPGDELIVGRTLY GCTFAFLHHGIGEFGVKIHHVDLNDAKALKAAINSKTRMIYFETPANPNMQLVDIAAVVE AVRGRDVLVVVDNTYCTPYLQRPLELGADLVVHSATKYLSGHGDITAGLVVGRKALVD RIRLEGLKDMTGAVLSPHDASLLMRGIKTLALRMDRHCANALQVAQFLARQPQVELIHY PGLPSFAQYELAQRQMRLPGGMIAFELKGGIDAGRRFMNALQLFARAVSLGDAESLAQH PASMTHS S YTPQERAHHGISEGLVRL S VGLED VEDLL AD VEQ ALQACKYP YD VPD YAEQ KLISEEDL (SEQ ID NO: 6) or variant thereof.

[0021] In certain embodiments, this disclosure relates to a recombinant vector disclosed herein wherein the nucleic acid sequence encoding the methioninase has the nucleotide sequence of: ATGTACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGTCAACA GCATGAGGGACTCACACAACAACACTGGCTTCTCTACCAGGGCCATCCATCATGGCT ACGATCCCCTGTCACATGGCGGAGCACTCGTGCCCCCTGTGTACCAGACCGCCACCT ACGCCTTCCCTACCGTGGAATATGGCGCCGCTTGCTTTGCCGGAGAGGAGGCCGGAC ACTTCTACAGCAGGATCTCTAATCCTACCCTGGCCCTGCTGGAGCAGCGGATGGCTTC TCTGGAGGGAGGCGAGGCTGGCCTGGCCCTGGCCTCTGGCATGGGCGCTATTACATC CACTATTTGGACTCTGCTGCGGCCTGGCGACGAGCTGATCGTGGGCAGGACCCTGTAT GGCTGCACTTTCGCTTTTCTGCACCACGGAATCGGCGAGTTTGGCGTGAAGATCCAC CACGTCGACCTCAATGATGCCAAAGCCCTGAAGGCTGCTATCAATTCTAAAACCAGA ATGATCTACTTCGAGACACCTGCTAATCCTAACATGCAGCTGGTGGATATCGCCGCCG TGGTGGAGGCCGTGAGAGGACGGGATGTGCTGGTCGTTGTGGACAATACATATTGCA CACCTTATCTGCAGCGGCCCCTGGAACTGGGCGCCGATCTGGTGGTGCACAGCGCAACCAAGTACCTGTCTGGGCATGGAGATATCACTGCCGGCCTGGTGGTGGGAAGAAAAG CTCTGGTTGACAGAATCCGTCTGGAGGGCCTGAAGGACATGACAGGAGCCGTGCTGA GTCCCCACGACGCCAGCCTGCTGATGAGGGGCATCAAGACCCTGGCTCTGAGAATGG ACAGGCACTGCGCAAATGCCCTCCAGGTCGCCCAGTTTCTGGCACGCCAGCCACAGG TGGAGCTGATCCACTACCCTGGCCTGCCATCCTTCGCCCAGTACGAGCTGGCTCAGA GACAGATGAGACTGCCTGGCGGTATGATTGCCTTTGAACTCAAGGGGGGCATCGACG CTGGAAGAAGATTCATGAACGCCCTGCAGCTGTTTGCCCGCGCTGTGTCACTGGGAG ACGCTGAGAGCCTGGCCCAGCACCCTGCCAGCATGACCCACTCCTCCTATACCCCTC AGGAGAGGGCTCACCACGGCATCAGCGAGGGCCTGGTGCGTCTGTCTGTGGGACTG GAGGATGTGGAGGACCTGCTGGCCGATGTGGAGCAGGCCCTGCAGGCTTGCAAGTA CCCATACGATGTTCCAGATTACGCTGAACAAAAACTCATCTCAGAAGAGGATCTGTGA

[0022] (SEQ ID NO: 7) or variants or degenerate codons encoding amino acids of a functional methioninase variant encoded therein.

[0023] In certain embodiments, this disclosure relates to a recombinant vector disclosed herein wherein the nucleic acid sequence encoding the methioninase and the pairs of loxP sites has the sequence of:

[0024] ATAACTTCGTATAGCATACATTATACGAAGTTATACTAGTGCTATCGATGTTGAGCTC AGGTACCTATAACTTCGTATAGGATACTTTATACGAAGTTATTCTAGAGCCACCATG TACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGTCAACAGCA TGAGGGACTCACACAACAACACTGGCTTCTCTACCAGGGCCATCCATCATGGCTACG ATCCCCTGTCACATGGCGGAGCACTCGTGCCCCCTGTGTACCAGACCGCCACCTACG CCTTCCCTACCGTGGAATATGGCGCCGCTTGCTTTGCCGGAGAGGAGGCCGGACACT TCTACAGCAGGATCTCTAATCCTACCCTGGCCCTGCTGGAGCAGCGGATGGCTTCTCT GGAGGGAGGCGAGGCTGGCCTGGCCCTGGCCTCTGGCATGGGCGCTATTACATCCAC TATTTGGACTCTGCTGCGGCCTGGCGACGAGCTGATCGTGGGCAGGACCCTGTATGG CTGCACTTTCGCTTTTCTGCACCACGGAATCGGCGAGTTTGGCGTGAAGATCCACCA CGTCGACCTCAATGATGCCAAAGCCCTGAAGGCTGCTATCAATTCTAAAACCAGAATG ATCTACTTCGAGACACCTGCTAATCCTAACATGCAGCTGGTGGATATCGCCGCCGTGG TGGAGGCCGTGAGAGGACGGGATGTGCTGGTCGTTGTGGACAATACATATTGCACAC CTTATCTGCAGCGGCCCCTGGAACTGGGCGCCGATCTGGTGGTGCACAGCGCAACCA AGTACCTGTCTGGGCATGGAGATATCACTGCCGGCCTGGTGGTGGGAAGAAAAGCTCTGGTTGACAGAATCCGTCTGGAGGGCCTGAAGGACATGACAGGAGCCGTGCTGAGT CCCCACGACGCCAGCCTGCTGATGAGGGGCATCAAGACCCTGGCTCTGAGAATGGAC AGGCACTGCGCAAATGCCCTCCAGGTCGCCCAGTTTCTGGCACGCCAGCCACAGGTG GAGCTGATCCACTACCCTGGCCTGCCATCCTTCGCCCAGTACGAGCTGGCTCAGAGA CAGATGAGACTGCCTGGCGGTATGATTGCCTTTGAACTCAAGGGGGGCATCGACGCT GGAAGAAGATTCATGAACGCCCTGCAGCTGTTTGCCCGCGCTGTGTCACTGGGAGAC GCTGAGAGCCTGGCCCAGCACCCTGCCAGCATGACCCACTCCTCCTATACCCCTCAG GAGAGGGCTCACCACGGCATCAGCGAGGGCCTGGTGCGTCTGTCTGTGGGACTGGA GGATGTGGAGGACCTGCTGGCCGATGTGGAGCAGGCCCTGCAGGCTTGCAAGTACCC ATACGATGTTCCAGATTACGCTGAACAAAAACTCATCTCAGAAGAGGATCTGTGACCT AGGATAACTTCGTATAATGTATGCTATACGAAGTTATCAAGCTTGCGTACGCGTGAC CGGTACTCGAGGATAACTTCGTATAAAGTATCCTATACGAAGTTAT (SEQ ID NO: 8) or variants or degenerate codons encoding amino acids of a functional methioninase encoded therein.

[0025] In certain embodiments, this disclosure relates to a recombinant vector disclosed herein comprising a TRE promoter, a start codon, and segments encoding a secretion sequence, a methioninase, and an HA sequence.

[0026] In certain embodiments, the recombinant vector further encodes a domain that specially binds a tumor specific antigen. In certain embodiments, the domain that specifically binds a tumor specific antigen is B7-H3 binding domain. In certain embodiments, the B7-H3 binding domain is linked to the N-terminal of methioninase and optionally HA is linked to the C-terminal of methioninase providing a fusion protein.

[0027] In certain embodiments, this disclosure relates to methods of treating cancer comprising administering a recombinant vector as discloses herein and an effective amount of Cre enzyme or nucleic acid / vector encoding a Cre enzyme to a subject in need thereof.

[0028] In certain embodiments, this disclosure relates to methods of treating cancer comprising administering a recombinant vector as disclosed herein, and transducing / transfecting cells ex vivo with vector encoding a Cre enzyme, or inserting a Cre enzyme into cells ex vivo, and administering an effective amount of the cells comprising the recombinant vector and / or Cre enzyme to a subject in need thereof.

[0029] In certain embodiments, the cells are macrophages, stem cells, immune cells, or other cells. In certain embodiments, the transduced cells are obtained from the subject (autologous) or notfrom the subject for treatment (allogeneic). In certain embodiments, transfecting is an inherent property of the vector or physical / mechanical transfection method, electroporation, sonoporation, magnetofection, gene microinjection, or laser irradiation.

[0030] In certain embodiments, this disclosure relates to methods of treating cancer comprising transducing cells ex vivo with Cre enzyme or vector encoding a Cre enzyme and a recombinant vector disclosed herein and administering an effective amount of the transduced cells to a subject in need thereof.

[0031] In certain embodiments, the cancer is a brain cancer, medulloblastoma, glioma, hematological cancer, solid tumor cancer, metastatic cancer, breast cancer, lung cancer, brain cancer, glioma, neuroblastoma, head or neck cancer, liver cancer, colon cancer, anal cancer, stomach cancer, pancreatic cancer, skin cancer, melanoma, prostate cancer, or ovarian cancer.

[0032] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0033] Figure 1 illustrates using bacterial methioninase (METase) to breakdown methionine. Methioninase is expressed from a vector and exposed to a Cre inducible enzyme thereby capable of expressing methioninase and degrading methionine, an essential amino acid necessary for protein synthesis in cancer cells. Methionine gemma lyase (methioninase or METase) is a bacterial enzyme that degrades methionine. Methionine is a nutrient needed by all eukaryotic cells; however, cancer cells have higher needs for methionine than normal cells. Constructs reported herein induce tumor cells or cells in the tumor microenvironment to express methioninase, depleting the tumor environment of methionine. The expression of methioninase may be regulated directly by Cre (Cre system) or indirectly by Cre inducible constructs, e.g., tetracycline / doxorubicin controlled systems (Tet system) to prevent or minimize harming normal cells.

[0034] Figure 2 illustrates a Cre inducible Murine Stem Cell Virus (MSCV) encoding a bacterial methioninase (Methionine gamma lyase gene). The recombinant virus is capable of inducing methioninase expression. A plasmid is used to generate MSCV viruses that can infect cancer cells. Cancer cells that are infected with this recombinant virus and also contain or express Cre will secrete methioninase due to the secretion sequence, i.e., the N-terminal IL2 secretion sequence. The reverse orientation loxP sites within the vector provides Cre-conditional controlled expression of methioninase. An antigen tag was included to facilitate detection. Green fluorescent protein(GFP) marker with nuclear localization (NLS) allows for identification of cells with viral transduction.

[0035] Figure 3 shows data indicating Cre plus either IL2-MET or IgK-MET cause METase expression (IgK-MET, detected intracellularly) and secretion (IL2-MET, detected extracellularly). Western blotting (immunoblotting, IB) was performed from cell lysates. Examples from a fibroblast cell line (3T3 cells - top two blots) and primary mouse cells from a medulloblastoma mouse model (GSMO cells - bottom two blots) are shown. The 1st blot IB:HA 3T3 lanes 1-4 indicate induction of IL2-METase and IgK-METase only when Cre enzyme is present to recombine the plasmid for METase expression. Lanes 5-8 are concentrated supernatant from cells showing secretion of both IL2 -METase and IgK-METase only when Cre enzyme is present. The 2nd blot from top 3T3 cells IB : b-tubulin is the loading control for intracellular blots showing even loading of the lanes for intracellular proteins. Right four lanes do not have b-tubulin as this is supernatant and not cell lysate. In the 3rd blot from the top GSMO IB:HA 3T3, lanes 1-4 indicate induction of IL2-METase and IgK-METase only when Cre enzyme is present to recombine the plasmid for METase expression. GSMO cells have endogenous Cre so instead of adding Cre like with 3T3 cells, cells were transduced with either mCherry as the control or the METase plasmid instead of doing both METase and Cre. The 4th blot from top GSMO cells IB: b-tubulin is the loading control for intracellular blots showing even loading of the lanes for intracellular proteins. Right four lanes do not have b-tubulin as this is supernatant and not cell lysate. This data indicates that the secretion sequence IL-2 sequence is optimal and the IgK sequence is also effective.

[0036] Figure 4 shows data when IL2-METase is switched on by Cre blocking the growth of human cell line HEK293.

[0037] Figure 5A illustrates a schematic of the plasmids used to generate methioninase in vitro and in vivo. TRE is Tet Responsive Element. TRE is used to inhibit inappropriate expression of methioninase. ATG is the start site of translation for the enzyme. IL2 is a secretion signal, thereby causing secretion of the enzyme. B7H3 binding domain is an antigen binding domain specific to the cancer cells such that one half of a recombinant protein will bind the cancer cell surface and the other half will degrade methionine extracellularly.

[0038] Figure 5B illustrates a Tet-on METase vector to be transduced into macrophages ex vivo optionally including a tumor specific binding agent. When the macrophages are injected in vivo, it is contemplated that macrophages home to tumors, e.g., via an antibody domain that recognizesa tumor specific antigen, e g., B7-H3 increasing the specificity of the anti-B7-H3-METase fusion protein.

[0039] Figure 5C illustrates a representation of how macrophages are transduced ex vivo to express METase once injected in vivo. A portion of the recombinant protein binds the surface of the tumor cell and the other half contains the METase enzyme to catalyzed degradation of extracellular methionine.

[0040] Figure 6 shows data directly testing METase efficiency using LC-MS technique showing that the design has optimal METase activity. Replicate wells of HEK293 cells were transfected with METase + Cre and compared to replicate controls transfected with Cre + mCherry (no METase controls) or METase without Cre (no Cre controls). The cells were incubated with Relabeled methionine, and LC-MS was use to quantify13C methionine in the cells. These data indicate that METase + Cre degrades methionine, while METase without Cre and Cre without METase do not. This provided evidence that METase construct works as to degrade methinonine effectively and under control of Cre.

[0041] Figure 7 show Kaplan-Meier curve data of time to tumor detection in RCAS DMG mice injected at P2 with METase or sham control. METase expression was induced in mouse with glioblastoma using RCAS virus as a vector for METase (RCAS-METase). The data indicates that RCAS-METase slowed tumor growth, prolonging mouse survival. This method induced METase expression lasting for weeks, without causing neurologic abnormalities or brain injury (100% of mice had normal neurologic exams).

[0042] DETAILED DISCUSSION

[0043] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims or as amended during prosecution.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein canalso be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0045] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0046] An "embodiment" of this disclosure refers to an example, but not necessarily limited to such example. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0047] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0048] As used herein, the term "about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to + / - a percentage of the specified value. In embodiments, about includes the specified value. In certain embodiments, the term “about” can include a 5 % or 10 % difference.

[0049] As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term “comprising” in reference to an oligonucleotide having a nucleic acid sequence refers to an oligonucleotide or peptide that may contain additional 5’ (5’ terminal end) or 3’ (3’ terminal end) nucleotides orN- or C-terminal aminoacids, i.e., the term is intended to include the oli onucleotide sequence or peptide sequence within a larger nucleic acid or peptide.

[0050] "Consisting essentially of or "consists of or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein that exclude certain prior art elements to provide an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel character! stic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein. The term “consisting of’ in reference to an oligonucleotide or peptide having a nucleotide or peptide sequence refers an oligonucleotide or peptide having the exact number of nucleotides or amino acids in the sequence and not more or having not more than a range of nucleotide expressly specified in the claim. For example, “5’ sequence consisting of’ is limited only to the 5’ end, i.e., the 3’ end may contain additional nucleotides. Similarly, a “3’ sequence consisting of’ is limited only to the 3’ end, and the 5’ end may contain additional nucleotides.

[0051] The terms, "nucleic acid," or "oligonucleotide," refer to a polymer of nucleotides, e.g., DNA, RNA, modified forms, or combinations thereof. The term "nucleotide" or its plural as used herein is interchangeable with modified forms as known in the art. In certain instances, the art uses the term "nucleobase" which embraces naturally-occurring nucleotide and non-naturally-occurring nucleotides which include modified nucleotides. Thus, nucleotide or nucleobase means the naturally occurring nucleobases A, G, C, T, and U and non-naturally occurring nucleobases, for example and without limitations, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N',N'-ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(Cs-Ce)-alkynyl-cytosine, 5-fluorouracil, 5 -bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-tr-iazolopyridin, isoguanine, and inosine. Methods of making oligonucleotides of a predetermined sequence are well-known. Solid-phase synthesis methods are preferred for both ribonucleotides and deoxyribonucleotides (the well-known methods of synthesizing DNA are also useful for synthesizing RNA). Ribonucleotides can also be prepared enzymatically.

[0052] The term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0053] The term "recombinant" when made in reference to a nucleic acid molecule refers to a nucleic acid molecule which is comprised of segments of nucleic acid joined together by means of molecular biological techniques provided that the entire nucleic acid sequence does not occurring in nature, i.e., there is at least one mutation in the overall sequence such that the entire sequence is not naturally occurring even though separately segments may occur in nature. The segments may be joined in an altered arrangement such that the entire nucleic acid sequence from start to finish does not naturally occur. The term "recombinant" when made in reference to a protein or a peptide refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.

[0054] The terms "vector" or " expression vector " refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free expression system. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. In certain embodiments, this disclosure contemplates a vector encoding a peptide disclosed herein in operable combination with a heterologous promoter.

[0055] In certain embodiments, the disclosure relates to the recombinant vectors comprising a nucleic acid encoding a polypeptide disclosed herein or fusion or chimeric protein thereof.

[0056] In certain embodiments, the recombinant vector optionally comprises a mammalian, human, insect, viral, bacterial, bacterial plasmid, yeast associated origin of replication or gene such as a gene or retroviral gene or lentiviral LTR, TAR, RRE, PE, SLIP, CRS, and INS nucleotide segment or gene selected from tat, rev, nef, vif, vpr, vpu, and vpx or structural genes selected from gag, pol, and env.

[0057] In certain embodiments, the recombinant vector optionally comprises a gene vector element (nucleic acid) such as a selectable marker region, lac operon, a CMV promoter, a hybridchicken B-actin / CMV enhancer (CAG) promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (IRES) sequence, cis-acting woodchuck post regulatory element (WPRE), scaffold-attachment region (SAR), inverted terminal repeats (ITR), FLAG tag coding region, c-myc tag coding region, metal affinity tag coding region, streptavidin binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 origin of replication, Col El origin of replication, fl origin, pBR322 origin, or pUC origin, TEV protease recognition site, loxP site, Cre recombinase coding region, or a multiple cloning site such as having 5, 6, or 7 or more restriction sites within a continuous segment of less than 50 or 60 nucleotides or having 3 or 4 or more restriction sites with a continuous segment of less than 20 or 30 nucleotides.

[0058] A “selectable marker” is a nucleic acid introduced into a recombinant vector that encodes a polypeptide that confers a trait suitable for artificial selection or identification (report gene), e.g., beta-lactamase confers antibiotic resistance, which allows an organism expressing beta-lactamase to survive in the presence antibiotic in a growth medium. Another example is thymidine kinase, which makes the host sensitive to ganciclovir selection. It may be a screenable marker that allows one to distinguish between wanted and unwanted cells based on the presence or absence of an expected color. For example, the lac-z-gene produces a beta-galactosidase enzyme which confers a blue color in the presence of X-gal (5-bromo-4-chloro-3-indolyl- -D-galactoside). If recombinant insertion inactivates the lac-z-gene, then the resulting colonies are colorless. There may be one or more selectable markers, e.g., an enzyme that can complement to the inability of an expression organism to synthesize a particular compound required for its growth (auxotrophic) and one able to convert a compound to another that is toxic for growth. URA3, an orotidine-51phosphate decarboxylase, is necessary for uracil biosynthesis and can complement ura3 mutants that are auxotrophic for uracil. URA3 also converts 5-fluoroorotic acid into the toxic compound 5 -fluorouracil. Additional contemplated selectable markers include any genes that impart antibacterial resistance or express a fluorescent protein. Examples include, but are not limited to, the following genes: ampr, camr, tetr, blasticidinr, neor, hygr, abxr, neomycin phosphotransferase type II gene (nptll), p-glucuronidase (gus), green fluorescent protein (gfp), egfp, yfp, mCherry, p-galactosidase (lacZ), lacZa, lacZAM15, chloramphenicol acetyltransferase (cat), alkaline phosphatase (phoA), bacterial luciferase (luxAB), bialaphos resistance gene (bar),phosphomannose isomerase (pmi), xylose isomerase (xylA), arabitol dehydrogenase (atlD), UDP-glucose:galactose-l-phosphate uridyltransferase (galT), feedback-insensitive a subunit of anthranilate synthase (OASA1D), 2-deoxy glucose (2-DOGR), benzyladenine-N-3 -glucuronide, E. coli threonine deaminase, glutamate 1 -semialdehyde aminotransferase (GSA-AT), D-amino acidoxidase (DAAO), salt-tolerance gene (rstB), ferredoxin-like protein (pflp), trehalose-6-P synthase gene (AtTPSl), lysine racemase (lyr), dihydrodipicolinate synthase (dapA), tryptophan synthase beta 1 (AtTSBl), dehalogenase (dhlA), mannose-6-phosphate reductase gene (M6PR), hygromycin phosphotransferase (HPT), and D-serine ammonialyase (dsdA).

[0059] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art.

[0060] A "variant" refers to a chemically similar nucleic acid or peptide sequence because of nucleic acid or amino acid changes. In certain embodiments, a variant contains one or two, or more nucleic acid or amino acid substitutions, deletions, or insertions. In certain embodiments, the substitutions are conserved substitutions. In certain embodiments, a variant contains one, two, or ten or more, or ten or less nucleic acid or amino acid additions. In certain embodiments, the additions may be to the 5 ’-terminus, 3 ’-terminus, N-terminus or the C-terminus. The variant may be substituted with one or more chemical substituents.

[0061] A conservative amino acid substitution refers to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. A variant may have "non-conservative" changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions (in other words, additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art. Variants can be tested in functional assays.

[0062] Contemplated variants include functional variants, allelic variants, or active fragments. Variants may include 1 or 2 amino acid substitutions or conserved substitutions. Variants may include 3 or 4 amino acid substitutions or conserved substitutions. Variants may include 5 or 6 or more amino acid substitutions or conserved substitutions. Variants include those with not more than 1% or 2% of the amino acids are substituted. Variants include those with not more than 3% or 4% of the amino acids are substituted. Variants include proteins with greater than 80%, 89%, 90%, 95%, 98%, or 99% identity or similarity.

[0063] Variants can be tested by mutating the vector to produce appropriate codon alternatives for polypeptide translation. Active variants and fragments can be identified with a high probability using computer modeling. Shihab et al. report an online genome tolerance browser. BMC Bioinformatics. 2017, 18( l):20. Ng et al. report methods of predicting the effects of amino acid substitutions on protein function. Annu Rev Genomics Hum Genet. 2006, 7:61-80. Teng et al. Approaches and resources for prediction of the effects of non-synonymous single nucleotide polymorphism on protein function and interactions. Curr Pharm Biotechnol. 2008, 9(2): 123-33.

[0064] Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, RaptorX, ESyPred3D, HHpred, Homology Modeling Professional for HyperChem, DNAStar, SPARKS-X, EVfold, Phyre, and Phyre2 software. See also Saldano et al. Evolutionary Conserved Positions Define Protein Conformational Diversity, PLoS Comput Biol. 2016, 12(3):el004775; Marks et al. Protein structure from sequence variation, Nat Biotechnol. 2012, 30(11): 1072-80; Mackenzie et al. Curr Opin Struct Biol. 2017, 44:161-167 Mackenzie et al. Proc Natl Acad Sci U S A. 113(47):E7438-E7447 (2016); Joseph et al. J R Soc Interface. 2014, 11(95):20131147, Wei et al. Int. J. Mol. Sci. 2016, 17(12), 2118. Variants can be tested in functional assays. Certain variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on).In certain embodiments, sequence "identity" refers to the number of exactly matching nucleic acids or amino acids (expressed as a percentage) in a sequence alignment between two sequences of the alignment calculated using the number of identical positions divided by the greater of the shortest sequence or the number of equivalent positions excluding overhangs wherein internal gaps are counted as an equivalent position. In certain embodiments, any recitation of sequence identity expressed herein may be substituted for sequence similarity.

[0065] Percent “similarity” is used to quantify the similarity between two sequences of the alignment. This method is identical to determining the identity except that certain amino acids do not have to be identical to have a match. Amino acids are classified as matches if they are among a group with similar properties according to the following amino acid groups: Aromatic - F Y W; hydrophobic-A V I L; Charged positive: R K H; Charged negative - D E; Polar - S T N Q. The amino acid groups are also considered conserved substitutions.

[0066] The term "specific binding agent" refers to a molecule, such as a proteinaceous molecule, which binds a target molecule with a greater affinity than other random molecules or proteins. Examples of specific binding agents include an antibody that bind an epitope of an antigen or a receptor which binds a ligand. In certain embodiments, "Specifically binds" refers to the ability of a specific binding agent (such as an ligand, receptor, enzyme, antibody or binding region / fragment thereof) to recognize and bind a target molecule or polypeptide, such that its affinity (as determined by, e.g., affinity ELISA or other assays) is at least 10 times as great, but optionally 50 times as great, 100, 250 or 500 times as great, or even at least 1000 times as great as the affinity of the same for any other or other random molecule or polypeptide.

[0067] In certain contexts, an “antibody” refers to a protein-based molecule that is naturally produced by animals in response to the presence of a protein or other molecule or that is not recognized by the animal’s immune system to be a “self’ molecule, i.e., recognized by the animal to be a foreign molecule, i.e., an antigen to the antibody. The immune system of the animal will create an antibody to specifically bind the antigen, and thereby targeting the antigen for degradation or elimination, or any cell or organism attached to the antigen. It is well recognized by skilled artisans that the molecular structure of a natural antibody can be synthesized and altered by laboratory techniques. Recombinant engineering can be used to generate fully synthetic antibodies or fragments thereof providing control over variations of the amino acid sequences of the antibody. Thus, the term “antibody” is intended to include natural antibodies, monoclonalantibody, or non-naturally produced synthetic antibodies, such as specific binding single chain antibodies, bispecific antibodies, or fragments thereof. These antibodies may have chemical modifications. The term "monoclonal antibodies" refers to a collection of antibodies encoded by the same nucleic acid molecule that are optionally produced by a single hybridoma (or clone thereof) or other cell line, or by a transgenic mammal such that each monoclonal antibody will typically recognize the same antigen. The term "monoclonal" is not limited to any particular method for making the antibody, nor is the term limited to antibodies produced in a particular species, e.g., mouse, rat, etc.

[0068] In humans, from a structural standpoint, an antibody is a combination of proteins: two heavy chain proteins and two light chain proteins. Alternatively, other animal produce antibodies from nucleic acids that encode a single protein. In humans, the heavy chains are longer than the light chains. The two heavy chains typically have the same amino acid sequence. Similarly, the two light chains typically have the same amino acid sequence. Each of the heavy and light chains contain a variable segment that contains amino acid sequences which participate in binding to the antigen. The variable segments of the heavy chain do not have the same amino acid sequences as the light chains. The variable segments are often referred to as the antigen binding domains. The antigen and the variable regions of the antibody may physically interact with each other at specific smaller segments of an antigen often referred to as the "epitope." Epitopes usually consist of surface groupings of molecules, for example, amino acids or carbohydrates. The terms “variable region,” "antigen binding domain," and "antigen binding region" refer to that portion of the antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. Small binding regions within the antigenbinding domain that typically interact with the epitope are also commonly alternatively referred to as the "complementarity-determining regions, or CDRs."

[0069] "Single chain antibodies" refer to a single peptide containing naturally or non-naturally occurring sequences, including synthetically modified peptide sequences, derived from an antibody variable region that specifically binds an antigen of interest. Single chain antibodies are sometimes fragments or variants of naturally occurring mammalian antibodies. Such antibodies are sometimes referred to as single-domain antibodies (sdAbs or VHHs), or camelid single-domain antibodies, e.g., when derived from an animal of Camelidae family, e.g., lamas, camels.A "heterologous" nucleic acid sequence or peptide sequence refers to a nucleic acid sequence or a peptide sequence that does not naturally occur, e.g., because the whole sequence contains a segment from other plants, bacteria, viruses, other organisms, or joinder of two sequences that occur the same organism but are j oined together in a manner that does not naturally occur in the same organism or any natural state.

[0070] Human macrophages are produced by the differentiation of monocytes in tissues and can be identified using flow cytometry or immunohistochemical staining by their specific expression of proteins such as CD14, CD40, CDllb, CD64, F4 / 80 (mice)ZEMRl (human), lysozyme M, MAC-l / MAC-3 and / or CD68.

[0071] Whole blood is composed of plasma, cells, immune cells, macrophages, red blood cells (RBCs; or erythrocytes), platelets, and nucleated white blood cells, also referred to as leukocytes. The leukocytes can be further categorized into mononuclear cells and polymorphonuclear cells (or granulocytes). There are different techniques to obtain peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells, leukocytes, or specific cell subsets, e.g., isolate specific cells directly by using flow cytometry, depleting red blood cells, centrifugation, and / or apheresis.

[0072] Certain cells, e.g., immune cells, can be purified and isolated from blood or bone marrow. For example, white blood cells are collected via apheresis, a process that withdraws blood from the body and removes one or more blood components (such as plasma, platelets, or other white blood cells). The remaining blood is then returned back into the body. The cells are exposed to a recombinant vector, which infects the cells in a way that a desired recombinant protein is produced.

[0073] Before and / or after infecting the isolated cells with the recombinant vector, the cells may be induced to replicate. The genetically modified cells may be expanded by growing cells in the laboratory until there are sufficient number of them. Optionally, these cells are frozen. The modified cells are then administered back to the patient. Various cell subsets, as well as T cell progenitors and other immune cells such as natural killer (NK) cells, macrophages, or monocytes can be induced to express a protein. Peripheral blood mononuclear cells (PBMCs) may be isolated by leukapheresis. T cells can be enriched by mononuclear cells counter-flow elutriation and expanded by addition of anti-CD3 / CD28 antibody coated paramagnetic beads for activation of T cells.Methods of Treating Cancer

[0074] Disclosed herein are nucleic acids and vectors encoding methioninase for uses in treating cancer. In certain embodiments, expression of methioninase is controlled using loxP sites and Cre enzymes allowing for cancer cell type targeting. In certain embodiments, this disclosure relates to method of treating cancer comprising administering an effective amount of a recombinant viral vector encoding methioninase in combination with administration of a Cre enzyme or nucleic acid or vector encoding the same to a subject in need thereof.

[0075] In certain embodiments, this disclosure relates to methods of treating cancer comprising administering an effective amount of Cre enzyme or nucleic acid / vector encoding a Cre enzyme and a recombinant vector encoding methioninase as discloses herein to a subject in need thereof.

[0076] In certain embodiments, this disclosure relates to methods of treating cancer comprising transducing cells ex vivo with Cre enzyme or vector encoding a Cre enzyme and a recombinant vector as disclosed herein encoding methioninase and administering an effective amount of the transduced cells to a subject in need thereof. In certain embodiments, the cells are macrophages.

[0077] In certain embodiments, the cancer is a brain cancer, medulloblastoma, glioma, hematological cancer, solid tumor cancer, metastatic cancer, breast cancer, lung cancer, brain cancer, neuroblastoma, head or neck cancer, liver cancer, colon cancer, anal cancer, stomach cancer, pancreatic cancer, skin cancer, melanoma, prostate cancer, or ovarian cancer.

[0078] In certain embodiments, the cancer is a solid tumor, cellular malignancy, or hematological malignancy such as leukemia, lymphoma, or multiple myeloma.

[0079] In certain embodiments, the cancer is ependymoma, lung cancer, non-small cell lung cancer, small cell lung cancer, bronchus cancer, mesothelioma, malignant pleural mesothelioma, lung adenocarcinoma, breast cancer, prostate cancer, colon cancer, rectum cancer, colorectal cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, cervical cancer, melanoma, kidney cancer, pancreatic cancer, pancreatic ductal adenocarcinoma (PDA), thyroid cancer, brain cancer, glioblastoma (GBM), medulloblastoma, glioma, neuroblastoma, liver cancer, bladder cancer, uterine cancer, bone cancer, osteosarcoma, sarcoma, rhabdomyosarcoma, Ewing's sarcoma, retinoblastoma, nasopharyngeal carcinoma.

[0080] In certain embodiments, a targeting sequence refers to any variety of polypeptide sequences capable of selectively binding to a targeted associated molecule. The targeting sequences may be derived from variable binding regions of antibodies, single chain antibodies,and antibody mimetics. In certain embodiments, targeting sequence is a single-chain variable fragment (scFv) derived from an antibody.

[0081] A "subject" refers to any animal, preferably a human patient, livestock, or domestic pet. As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments of the present disclosure also contemplate treatment that merely reduces symptoms, and / or delays disease progression.

[0082] As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

[0083] As used herein, the term "combination with" when used to describe administration of an agent with an additional treatment means such that the agent may be administered prior to, together with, or after the additional treatment, or a combination thereof, such that multiple agents are pharmacologically available at some overlapping time, e.g. considering the half-life of each agent.

[0084] The term "effective amount" or "therapeutically effective amount" refers to that amount of a compound or pharmaceutical composition described herein that is sufficient to effect the intended application including, but not limited to, disease treatment, as illustrated below. The therapeutically effective amount can vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.

[0085] "Cancer" refers any of various cellular diseases with malignant neoplasms characterized by the proliferation of cells. It is not intended that the diseased cells must actually invade surrounding tissue and metastasize to new body sites. Cancer can involve any tissue of the body and have many different forms in each body area. Within the context of certain embodiments, whether "cancer is reduced" may be identified by a variety of diagnostic manners known to one skill in the art including, but not limited to, observation the reduction in size or number of tumor masses or if an increase of apoptosis of cancer cells observed, e.g., if more than a 5 % increase in apoptosis of cancer cells is observed for a sample compound compared to a control without the compound. It may also be identified by a change in relevant biomarker or gene expression profile, such as PSA for prostate cancer, HER2 for breast cancer, or others.The cancer to be treated in the context of the present disclosure may be any type of cancer or tumor. These tumors or cancer include, and are not limited to, tumors of the hematopoietic and lymphoid tissues or hematopoietic and lymphoid malignancies, tumors that affect the blood, bone marrow, lymph, and lymphatic system. Hematological malignancies may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines. The myeloid cell line normally produces granulocytes, erythrocytes, thrombocytes, macrophages and mast cells; the lymphoid cell line produces B, T, NK and plasma cells. Lymphomas, lymphocytic leukemias, and myeloma are from the lymphoid line, while acute and chronic myelogenous leukemia, tri coleucemia / hairy cell leukemia (HCL), myelodysplastic syndromes and myeloproliferative diseases are myeloid in origin.

[0086] Also contemplated are malignancies located in the colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, hypophysis, testicles, ovaries, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax and genitourinary apparatus and, more particularly, childhood acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult (primary) hepatocellular cancer, adult (primary) liver cancer, adult acute lymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin's disease, adult Hodgkin's lymphoma, adult lymphocytic leukemia, adult non-Hodgkin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignant tumors, anal cancer, astrocytoma, cancer of the biliary tract, cancer of the bladder, bone cancer, brain stem glioma, brain tumors, breast cancer, cancer of the renal pelvis and ureter, primary central nervous system lymphoma, central nervous system lymphoma, cerebellar astrocytoma, brain astrocytoma, cancer of the cervix, childhood (primary) hepatocellular cancer, childhood (primary) liver cancer, childhood acute lymphoblastic leukemia, childhood acute myeloid leukemia, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood brain astrocytoma, childhood extracranial germ cell tumors, childhood Hodgkin's disease, childhood Hodgkin's lymphoma, childhood visual pathway and hypothalamic glioma, childhood lymphoblastic leukemia, childhood medulloblastoma, childhood non-Hodgkin's lymphoma, childhood supratentorial primitive neuroectodermal and pineal tumors, childhood primary liver cancer, childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhood visual pathway and hypothalamic glioma, chronic lymphocytic leukemia, chronic myeloidleukemia, cancer of the colon, cutaneous T-cell lymphoma, endocrine pancreatic islet cells carcinoma, endometrial cancer, ependymoma, epithelial cancer, cancer of the esophagus, Ewing's sarcoma and related tumors, cancer of the exocrine pancreas, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic biliary tract cancer, cancer of the eye, breast cancer in women, Gaucher's disease, cancer of the gallbladder, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal tumors, germ cell tumors, gestational trophoblastic tumor, head and neck cancer, hepatocellular cancer, Hodgkin's disease, Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancers, intraocular melanoma, islet cell carcinoma, islet cell pancreatic cancer, Kaposi's sarcoma, cancer of kidney, cancer of the larynx, cancer of the lip and mouth, cancer of the liver, cancer of the lung, lymphoproliferative disorders, macroglobulinemia, breast cancer in men, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, occult primary metastatic squamous neck cancer, primary metastatic squamous neck cancer, metastatic squamous neck cancer, multiple myeloma, multiple myeloma / plasmatic cell neoplasia, myelodysplastic syndrome, myelogenous leukemia, myeloid leukemia, myeloproliferative disorders, paranasal sinus and nasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma during pregnancy, nonmelanoma skin cancer, non-small cell lung cancer, metastatic squamous neck cancer with occult primary, buccopharyngeal cancer, malignant fibrous histiocytoma, malignant fibrous osteosarcoma / histiocytoma of the bone, epithelial ovarian cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, paraproteinemias, purpura, parathyroid cancer, cancer of the penis, phaeochromocytoma, hypophysis tumor, neoplasia of plasmatic cells / multiple myeloma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell cancer, cancer of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, cancer of the salivary glands, sarcoidosis, sarcomas, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous neck cancer, stomach cancer, pineal and supratentorial primitive neuroectodermal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, transitional renal pelvis and ureter cancer, trophoblastic tumors, cell cancer of the renal pelvis and ureter, cancer of the urethra, cancer of the uterus, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic glioma, cancer of the vulva, Waldenstrom's macroglobulinemia, Wilms' tumor and any otherhyperproliferative disease, as well as neoplasia, located in the system of a previously mentioned organ.

[0087] In certain embodiments, constructs disclosed herein are designed to bind a tumor or cancer specific antigen such as B7-H3 antigen (CD276). Other contemplated antigens include cluster of differentiation 19 (CD 19), cluster of differentiation 10 (CD 10), cluster of differentiation 20 (CD20), cluster of differentiation 33 (CD33), cluster of differentiation 38 (CD38), CD70 (tumor necrosis factor ligand superfamily member 7), CD133 (prominin 1), CD171 (LI cell adhesion molecule), (EGFR) epidermal growth factor receptor, (HER2) human epidermal growth factor receptor 2, EGFR vIII (epidermal growth factor receptor variant 3) (MUC1) mucinl, (MUC16) mucin 16, (EpCAM) epithelial cell adhesion molecule, (AFP) alpha-fetoprotein, (FAP) familial adenomatous polyposis, (CEA) carcinoembryonic antigen, (PSCA) prostate stem cell antigen, (PSMA) prostate-specific membrane antigen, (PSA) prostate-specific antigen, (AXL) AXL receptor tyrosine kinase, (DLL3) delta-like 3, (EPHA2) EPH receptor A2, (FRa) folate receptor alpha, (LMP1) Epstein-Barr virus latent membrane protein 1, (MAGE) melanoma antigen gene protein, MAGE-A1, MAGE-A3, MAGE-A4, (DR5) death receptor 5, (NKG2D) natural killer group 2 member D receptor, (CAIX) carbonic anhydrase IX, (TAG-72) tumor-associated glycoprotein 72, (GUCY2C) guanylate cyclase 2C, (ANTXR1) anthrax toxin receptor 1, (GSPG4) general secretion pathway protein G, (ROR) RAR-related orphan receptors, ROR1 (receptor tyrosine kinase like orphan receptor 1), IL13RA2 (Interleukin 13 Receptor Subunit Alpha 2), Wilms' tumor 1 (WT1), Survivin, Tn (aGalNAc-O-Ser / Thr), sialyl-Tn (aNeuAc2,6-aGalNAc-O-Ser / Thr), TF (bGall,3-aGalNAc-O-Ser / Thr), CA 19-9 (Neu5Aca2-3Gaipi-3[Fucal-4]GlcNAcP), Telomerase reverse transcriptase (TERT), Beta-hCG (Human chorionic gonadotropin), p53, Ras, bladder tumor antigen (BTA), antibody specific antigen Om5, GD2 (Ganglioside GD2), integrin alpha- v / beta-6, mesothelin antigen, BCMA (TNF receptor superfamily member 17\B-cell maturation protein), CD123 (interleukin 3 receptor subunit a\CD123 antigen), CD138 (syndecan 1), CD22 (SIGLEC2), CD5 (lymphocyte antigen Tl / Leu-1), Ig kappa chain, LeY (fucosyltransferase 3 / Lewis Blood Group), NKG2D ligand (killer cell lectin like receptor K1 / CD314), WT1 (Wilms’ tumor antigen 1), C-Met (MET proto-oncogene), CAIX (carbonic anhydrase 9), GPC3 (glypican 3), HPV16-E6 (human papillomavirus E6 protein), MARTI (melan-A), NY-ESO-1 (cancer / testis antigen IB), PD-L1 (CD274 molecule), PSMA (folate hydrolase 1), or VEGFR2 (kinase insert domain receptor / vascular endothelial growth factor receptor 2).In certain embodiments, methods disclosed herein are used in combination with surgery, e.g., to remove a tumor or cancerous tissue, with radiation and / or chemotherapy.

[0088] In certain embodiments, this disclosure contemplates that constructs disclosed herein can be used to treat cancer in combination with another anti-cancer agent. A “chemotherapy agent,” “chemotherapeutic,” “anti-cancer agent,” or the like, refer to molecules that are recognized to aid in the treatment of a cancer. Contemplated examples include the following molecules or derivatives such as abemaciclib, abiraterone acetate, methotrexate, paclitaxel, adriamycin, acalabrutinib, brentuximab vedotin, ado-trastuzumab emtansine, aflibercept, afatinib, netupitant, palonosetron, imiquimod, aldesleukin, alectinib, alemtuzumab, pemetrexed disodium, copanlisib, melphalan, brigatinib, chlorambucil, amifostine, aminolevulinic acid, anastrozole, apalutamide, aprepitant, pamidronate disodium, exemestane, nelarabine, arsenic trioxide, ofatumumab, atezolizumab, bevacizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, carmustine, belinostat, bendamustine, inotuzumab ozogamicin, bevacizumab, bexarotene, bicalutamide, bleomycin, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, brigatinib, busulfan, irinotecan, capecitabine, fluorouracil, carboplatin, carfilzomib, ceritinib, daunorubicin, cetuximab, cisplatin, cladribine, cyclophosphamide, clofarabine, cobimetinib, cabozantinib-S-malate, dactinomycin, crizotinib, ifosfamide, ramucirumab, cytarabine, dabrafenib, dacarbazine, decitabine, daratumumab, dasatinib, defibrotide, degarelix, denileukin diftitox, denosumab, dexamethasone, dexrazoxane, dinutuximab, docetaxel, doxorubicin, durvalumab, rasburicase, epirubicin, elotuzumab, oxaliplatin, eltrombopag olamine, enasidenib, enzalutamide, eribulin, vismodegib, erlotinib, etoposide, everolimus, raloxifene, toremifene, panobinostat, fulvestrant, letrozole, filgrastim, fludarabine, flutamide, pralatrexate, obinutuzumab, gefitinib, gemcitabine, gemtuzumab ozogamicin, glucarpidase, goserelin, propranolol, trastuzumab, topotecan, palbociclib, ibritumomab tiuxetan, ibrutinib, ponatinib, idarubicin, idelalisib, imatinib, talimogene laherparepvec, ipilimumab, romidepsin, ixabepilone, ixazomib, ruxolitinib, cabazitaxel, palifermin, pembrolizumab, ribociclib, tisagenlecleucel, lanreotide, lapatinib, olaratumab, lenalidomide, lenvatinib, leucovorin, leuprolide, lomustine, trifluridine, olaparib, vincristine, procarbazine, mechlorethamine, megestrol, trametinib, temozolomide, methylnaltrexone bromide, midostaurin, mitomycin C, mitoxantrone, plerixafor, vinorelbine, necitumumab, neratinib, sorafenib, nilutamide, nilotinib, niraparib, nivolumab, tamoxifen, romiplostim, sonidegib, omacetaxine, pegaspargase, ondansetron, osimertinib, panitumumab, pazopanib, interferon alfa-2b, pertuzumab, pomalidomide, mercaptopurine, regorafenib, rituximab, rolapitant, rucaparib, siltuximab, sunitinib, thioguanine, temsirolimus, thalidomide, thiotepa, trabectedin, valrubicin, vandetanib, vinblastine, vemurafenib, vorinostat, zoledronic acid, or combinations thereof such as cyclophosphamide, methotrexate, 5 -fluorouracil (CMF); doxorubicin, cyclophosphamide (AC); mustine, vincristine, procarbazine, prednisolone (MOPP); adriamycin, bleomycin, vinblastine, dacarbazine (ABVD); cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP); bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin, 5 -fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX); methotrexate, vincristine, doxorubicin, cisplatin (MVAC).

[0089] In certain embodiments, the chemotherapy agent is an anti-PD-1, anti-PD-Ll anti-CTLA4 antibody or combinations thereof, such as an anti-CTLA4 (e.g., ipilimumab, tremelimumab) and anti-PDl (e.g., nivolumab, pembrolizumab, cemiplimab) and anti-PD-Ll (e.g., atezolizumab, avelumab, durvalumab).

[0090] In certain embodiments, the method of administration is in a subject with a lymphodepleted environment due to prior or concurrent administration of lymphodepleting agents. In certain embodiments, lymphodepleting agents (e.g., cyclophosphamide and fludarabine).

[0091] Pharmaceutical compositions

[0092] The present disclosure contemplates that a pharmaceutical agent(s) can be administered to a subject either alone or as a part of a pharmaceutical composition. In certain embodiments, this disclosure relates to pharmaceutical compositions and kits comprising nucleic acids and vectors encoding methioninase and / or a Cre enzyme or vector encoding the same, i.e., as pharmaceutical agents.

[0093] Pharmaceutical compositions typically comprise an effective amount of a pharmaceutical agent(s) and a suitable pharmaceutical acceptable excipient or carrier. The preparations can be prepared in a manner known per se, which usually involves mixing the pharmaceutical agent(s) according to the disclosure with the one or more pharmaceutically acceptable excipients / carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary, under aseptic conditions.

[0094] In certain embodiments, this disclosure contemplates an intravenous formulation with pH buffering agents and tonicity in a range representing physiological values (pH 7 to 8) or for bolus administration, e.g., containing normal saline or dextrose optionally containing pH bufferingagents. In certain embodiments, the pharmaceutical composition is in the form of a sterilized pH buffered aqueous salt solution or a saline phosphate buffer between a pH of 6 to 8, optionally comprising a saccharide or polysaccharide.

[0095] Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable (such as olive oil, sesame oil) and injectable organic esters such as ethyl oleate.

[0096] In certain embodiments, this disclosure relates to pharmaceutical compositions and kits comprising nucleic acids and vectors encoding methioninase and / or a Cre enzyme or vector encoding a Cre enzyme comprising a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutically acceptable excipient is selected from lactose, sucrose, mannitol, triethyl citrate, dextrose, cellulose, methyl cellulose, ethyl cellulose, hydroxyl propyl cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, croscarmellose sodium, polyvinyl N-pyrrolidone, crospovidone, ethyl cellulose, povidone, methyl and ethyl acrylate copolymer, polyethylene glycol, fatty acid esters of sorbitol, lauryl sulfate, gelatin, glycerin, glyceryl monooleate, silicon dioxide, titanium dioxide, talc, com starch, carnauba wax, stearic acid, sorbic acid, magnesium stearate, calcium stearate, castor oil, mineral oil, calcium phosphate, starch, carboxymethyl ether of starch, iron oxide, triacetin, acacia gum, esters, or salts thereof.

[0097] These compositions may also contain preserving, emulsifying, and dispensing agents. Prevention of the action of microorganisms may be controlled by addition of any of various antibacterial and antifungal agents, example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0098] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, com germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.

[0099] The pharmaceutical compositions of the present disclosure can be administered to subjects either parenterally (directly into a tumor or an area or organ of a subject containing cancerous cells, intravenously, intramuscularly, or subcutaneously), orally, intracistemally, intravaginally, intraperitoneally, intravesically, rectally, locally (powders, ointments, or drops), topically to the skin, or as a buccal or nasal spray.

[0100] The pharmaceutical preparations of the disclosure are preferably in a unit dosage form, and can be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which can be properly labeled); optionally with one or more leaflets containing product information and / or instructions for use.

[0101] In certain embodiments, this disclosure contemplates a kit comprising pharmaceutical agents disclosed herein and a container with a suitable diluent. Further components of the kit may be instructions for use, administration means, such as syringes, catheters, brushes, etc. (if the compositions are not already provided in the administration means) or other components necessary for use in medical (surgical) practice, such as substitute needles or catheters, extra vials or further wound cover means. In certain embodiments, the kit comprises a syringe housing the dry and stable hemostatic composition and a syringe containing the diluent (or provided to take up the diluent from another diluent container).

[0102] Methioninase inducing virus for anti-cancer therapy

[0103] In certain embodiments, a recombinant virus that induces expression of the bacterial enzyme methioninase in mammalian cells, for the purpose of treating brain tumors. Methionine is an enzyme that degrades the amino acid methionine, and this enzyme is not expressed in mammalian cells, due to the importance of methionine for mammalian biology. Experiments indicate that the pediatric brain tumor called medulloblastoma depends on robust supply of methionine, and that dietary methionine deprivation impairs tumor growth. To induce stronger methionine deprivation than can be achieved through diet, and to limit methionine deprivation to the tumor microenvironment, recombinant retroviral constructs were designed that can transduce cells to express and secrete bacterial methioninase. the DNA sequence of pseudomonasmethioninase was altered by optimizing codon utilization while preserving the encoded amino acid sequence. A variety of secretion signal sequences were added 5' to the methioninase sequence and a tag (Myc) was added to the 3' end to facilitate detection of the expressed protein. The coding region was placed into Cre-Conditional MSCV and lentiviral plasmids, using the requirement for Cre expression as a vital safety switch to prevent more widespread methioninase expression than intended. These plasmids were used to generate MSCV and lentivirus particles that transduce mammalian cells. Methioninase inducing viruses were tested, and Cre-conditional expression and secretion of methioninase protein was verified. Importantly, these viruses stopped the growth of cancer cells.

[0104] In certain embodiments, this disclosure relates to Cre-inducible viral constructs that selectively expresses methionine degrading enzyme (methioninase) for treatment of tumors, e.g., brain tumors. Methionine is an essential amino acid. Many tumors are addicted to methionine. Methionine gemma lyase (aka methioninase or METase) is a bacterial enzyme that degrades methionine. This enzyme is not identified as be expressed in mammalian cells. Figure 1 shows a reaction schematic showing how L-Methionine, the amino acid required to initiate translation of any protein, can be degraded by methioninase into metabolites.

[0105] Medulloblastoma (MB), a tumor of the cerebellum, is the most frequent brain cancer in childhood and a major cause of pediatric mortality. MB depends on a robust supply of methionine. Dietary methionine deprivation impairs tumor growth. However, a stronger methionine deprivation in the local tumor environment is needed.

[0106] Disclosed herein are recombinant retroviral constructs that can transduce cells to express and secrete bacterial methioninase, thereby inducing tumor cells or cells in the tumor microenvironment to express methioninase, depleting the tumor environment of methionine. In certain embodiments, the construct has the following sequence (wherein the loxP cites are in bold):

[0107] TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGT AACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTA GGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGT TACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGAC GATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGG GTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTA TAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCA GGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCC TTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAAC CGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGC AGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCC CGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGC GGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGC TTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTC ACAATTCTCCCCAGCTCTTTCTGCGCTGTCCATAATGAACTATTTCAGGAGTATTGGAG TATGTGGATTCAAAACAGTGGCTAATTGAGTCAGGATTACTTAATATTCGACTCCTACC CTCAGAATGTTGTTCTCCAACTTCAAGAAACTGTTACCCATGAAAGACCCCCGCTGA CGGGTAGTCAATCACTCAGAGGAGACCCTCCCAAGGAACAGCGAGACCACAAGTCG GATGCAACTGCAAGAGGGTTTATTGGATACACGGGTACCCGGGCGACGCAGTCTATC GGAGGACTGGCGCGCCGAGTGAGGGGTTGTGGGCTCTTTTATTGAGCTCGGGGAGCA GAAGCGCGCGAACAGAAGCGAGAAGCGAACTGATTGGTTAGTTCAAATAAGGCACA GGGTCATTTCAGGTCCTTGGGGCACCCTGGAAACATCTGATGGTTCTCTAGAAACTGC TGAGGGCGGGACCGCATCTGGGGACCATCTGTTCTTGGCCCTGAGCCGGGGCAGGA ACTGCTTACCACAGATATCCTGTTTGGCCCATATTCTGCTGTCTCTCTGTTCCTAACCTT GATCTGAACTTCTCTATTCTCAGTTATGTATTTTCCATGCCTTGCAAAATGGCGTTACTT AAGCTAGCTTGCCAAACCTACAGGTGGGGTCTTTCATTCCCCCCTTTTTCTGGAGACT AAATAAAATCTTTTATTTTATCGATAAGCTTGGCTGCAGGTCGACGGATCTCTCGAGCC ACTTTGTACAAGAAAGCTGGGTCCGATGCGGGGAGGCGGCCCAAAGGGAGATCCGA CTCGTCTGAGGGCGAAGGCGAAGACGCGGAAGAGGCCGCAGAGCCGGCAGCAGGC CGCGGGAAGGAAGGTCCGCTGGATTGAGGGCCGAAGGGACGTAGCAGAAGGACGTC CCGCGCAGAATCCAGGTGGCAACACAGGCGAGCAGCCATGGAAAGGACGTCAGCTT CCCCGACAACACCACGGAATTGTCAGTGCCCAACAGCCGAGCCCCTGTCCAGCAGC GGGCAAGGCAGGCGGCGATGAGTTCCGCCGTGGCAATAGGGAGGGGGAAAGCGAA AGTCCCGGAAAGGAGCTGACAGGTGGTGGCAATGCCCCAACCAGTGGGGGTTGCGT CAGCAAACACAGTGCACACCACGCCACGTTGCCTGACAACGGGCCACAACTCCTCATAAAGAGACAGCAACCAGGATTTATACAAGGAGGAGAAAATGAAAGCCATACGGGA AGCAATAGCATGATACAAAGGCATTAAAGCAGCGTATCCACATAGCGTAAAAGGAGC AACATAGTTAAGAATACCAGTCAATCTTTCACAAATTTTGTAATCCAGAGGTTGATTAT CGTTACTTGTACAGCTCGTCCATGCCGCCGGTGGAGTGGCGGCCCTCGGCGCGTTCG TACTGTTCCACGATGGTGTAGTCCTCGTTGTGGGAGGTGATGTCCAACTTGATGTTGA CGTTGTAGGCGCCGGGCAGCTGCACGGGCTTCTTGGCCTTGTAGGTGGTCTTGACCT CAGCGTCGTAGTGGCCGCCGTCCTTCAGCTTCAGCCTCTGCTTGATCTCGCCCTTCAG GGCGCCGTCCTCGGGGTACATCCGCTCGGAGGAGGCCTCCCAGCCCATGGTCTTCTT CTGCATTACGGGGCCGTCGGAGGGGAAGTTGGTGCCGCGCAGCTTCACCTTGTAGAT GAACTCGCCGTCCTGCAGGGAGGAGTCCTGGGTCACGGTCACCACGCCGCCGTCCTC GAAGTTCATCACGCGCTCCCACTTGAAGCCCTCGGGGAAGGACAGCTTCAAGTAGTC GGGGATGTCGGCGGGGTGCTTCACGTAGGCCTTGGAGCCGTACATGAACTGAGGGGA CAGGATGTCCCAGGCGAAGGGCAGGGGGCCACCCTTGGTCACCTTCAGCTTGGCGG TCTGGGTGCCCTCGTAGGGGCGGCCCTCGCCCTCGCCCTCGATCTCGAACTCGTGGC CGTTCACGGAGCCCTCCATGTGCACCTTGAAGCGCATGAACTCCTTGATGATGGCCAT GTTATCCTCCTCGCCCTTGCTCACCATGGTGGCGCAGTGGGTTCTCTAGTTAGCCAGA GAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGG GGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACT CCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCC ACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACG TAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGG CGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTG ATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAA GTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTC GTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCGGAACTCCATA TATGGGCTATGAACTAATGACCCCGTAATTGATTACTATTAATAACTAGTCAATAATCAA TGTCAACATAACTTCGTATAGCATACATTATACGAAGTTATACTAGTGCTATCGATGT TGAGCTCAGGTACCTATAACTTCGTATAGGATACTTTATACGAAGTTATTCTAGAGC CACCATGTACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGTC AACAGCATGAGGGACTCACACAACAACACTGGCTTCTCTACCAGGGCCATCCATCAT GGCTACGATCCCCTGTCACATGGCGGAGCACTCGTGCCCCCTGTGTACCAGACCGCCACCTACGCCTTCCCTACCGTGGAATATGGCGCCGCTTGCTTTGCCGGAGAGGAGGCC GGACACTTCTACAGCAGGATCTCTAATCCTACCCTGGCCCTGCTGGAGCAGCGGATGG CTTCTCTGGAGGGAGGCGAGGCTGGCCTGGCCCTGGCCTCTGGCATGGGCGCTATTA CATCCACTATTTGGACTCTGCTGCGGCCTGGCGACGAGCTGATCGTGGGCAGGACCC TGTATGGCTGCACTTTCGCTTTTCTGCACCACGGAATCGGCGAGTTTGGCGTGAAGAT CCACCACGTCGACCTCAATGATGCCAAAGCCCTGAAGGCTGCTATCAATTCTAAAACC AGAATGATCTACTTCGAGACACCTGCTAATCCTAACATGCAGCTGGTGGATATCGCCG CCGTGGTGGAGGCCGTGAGAGGACGGGATGTGCTGGTCGTTGTGGACAATACATATT GCACACCTTATCTGCAGCGGCCCCTGGAACTGGGCGCCGATCTGGTGGTGCACAGCG CAACCAAGTACCTGTCTGGGCATGGAGATATCACTGCCGGCCTGGTGGTGGGAAGAA AAGCTCTGGTTGACAGAATCCGTCTGGAGGGCCTGAAGGACATGACAGGAGCCGTG CTGAGTCCCCACGACGCCAGCCTGCTGATGAGGGGCATCAAGACCCTGGCTCTGAGA ATGGACAGGCACTGCGCAAATGCCCTCCAGGTCGCCCAGTTTCTGGCACGCCAGCCA CAGGTGGAGCTGATCCACTACCCTGGCCTGCCATCCTTCGCCCAGTACGAGCTGGCTC AGAGACAGATGAGACTGCCTGGCGGTATGATTGCCTTTGAACTCAAGGGGGGCATCG ACGCTGGAAGAAGATTCATGAACGCCCTGCAGCTGTTTGCCCGCGCTGTGTCACTGG GAGACGCTGAGAGCCTGGCCCAGCACCCTGCCAGCATGACCCACTCCTCCTATACCC CTCAGGAGAGGGCTCACCACGGCATCAGCGAGGGCCTGGTGCGTCTGTCTGTGGGA CTGGAGGATGTGGAGGACCTGCTGGCCGATGTGGAGCAGGCCCTGCAGGCTTGCAA GTACCCATACGATGTTCCAGATTACGCTGAACAAAAACTCATCTCAGAAGAGGATCTG TGACCTAGGATAACTTCGTATAATGTATGCTATACGAAGTTATCAAGCTTGCGTACGC GTGACCGGTACTCGAGGATAACTTCGTATAAAGTATCCTATACGAAGTTATAGCCTGCTTTTTTGTACAAACTTGGAATTCCGGCGCCTAGAGAAGGAGTGAGGGCTGGATAAAGGGAGGATCGAGGCGGGGTCGAACGAGGAGGTTCAAGGGGGAGAGACGGGGCGGA TGGAGGAAGAGGAGGCGGAGGCTTAGGGTGTACAAAGGGCTTGACCCAGGGAGGG GGGTCAAAAGCCAAGGCTTCCCAGGTCACGATGTAGGGGACCTGGTCTGGGTGTCCA TGCGGGCCAGGTGAAAAGACCTTGATCTTAACCTGGGTGATGAGGTCTCGGTTAAAG GTGCCGTCTCGCGGCCATCCGACGTTAAAGGTTGGCCATTCTGCAGAGCAGAAGGTA ACCCAACGTCTCTTCTTGACATCTACCGACTGGTTGTGAGCGATCCGCTCGACATCTT TCCAGTGACCTAAGGTCAAACTTAAGGGAGTGGTAACAGTCTGGCCCTAATTTTCAG ACAAATACAGAAACACAGTCAGACAGAGACAACACAGAACGATGCTGCAGCGCTGCAGCAGACAAGACGCGCGGCTTCGGTTCCAAACCGAAAGCAAAAATTCAGACGGAGG CGGGAACTGTTTTAGGTTCTCGTCTCCTACCAGAACCACATATCCTGACGGGGTCGGA TTCCACATCGACTCCCTTCCTCAGGTCGGGCCACAAAAACGGCCCCCAAAGTCCCTG GGACGTCTCCCAGGGTTGCGGCCGGGTGTTCAGAACTCGTCAGTTCCACCACGGGTC CGCCAGATACAGAGCTAGTTAGCTAACTAGTACAGACGCAGGCGCAAACATTAGATG CCGGCACAAACACGCACAAAGACAGAGACAGACACGAAACGACCGCTGGCCAGCT TACCTCCCGGCGGGGGGGTCGGTGGTCCCTGGGCAGGGGTCTCCAAATCTCGGTGGA ACCTCCAAATGAAAGACCCCCGAGGTGGGCAGTCAATCAATCTGAGGAGACCCTCCC AAGGATCAGCGAGTCCACGATTCGGATGCAAACAGCAAGAGGCTTTATTGGGAATAC GGGTACCCGGGCGACGCAGTCTATCGGAGGACTGGCGCGCCGAGTGAGGGGTTGTG GGCTCTTTTATTGAGCTCGGGGAGCAGAAGCGCGCGAACAGAAGCGAGAAGCGAAC TGATTGGTTAGTTCAAATAAGGCACAGGGTCATTTCAGGTCCTTGGGGCACCCTGGA AACATCTGATGGTTCTCTAGAAACTGCTGAGGGCGGGACCGCATCTGGGGACCATCT GTTCTTGGCCCTGAGCCGGGGCAGGAACTGCTTACCACAGATATCCTGTTTGGCCCAT ATTCTGCTGTCTCTCTGTTCCTAACCTTGATCTGAACTTCTCTATTCTCAGTTATGTATT TTCCATGCCTTGCAAAATGGCGTTACTTAAGCTAGCTTGCCAAACCTACAGGTGGGGT CTTTCATTCCCCCCTTTTTCTGGAGACTAAATAAAATCTTTTATTTTATCTATGGCTCGT ACTCTATAGGCTTCAGCTGGTGATATTGTTGAGTCAAAACTAGAGCCTGGACCACTGA TATCCTGTCTTTAACAAATTGGACTAATCGCCTCTACGCCGGACGCATCGTGGCCGGC ATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGG GAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTG GCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTT GCGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATC GCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCG ATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTT TCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCT GCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGC CCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCG GGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAG GGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACG TCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTG AAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCG GCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTG AAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGA TCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCT GCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGC ATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTA CGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACAC TGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTT GCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGA AGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTT GCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGAC TGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGC TGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAG CACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCA GGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAA GCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTC ATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATC CCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGA TCTTC (SEQ ID NO: 4) or variants or degenerate codon usage.

[0108] In certain embodiments, this disclosure relates to a recombinant viral vector comprising a segment encoding methioninase and two pairs of loxP sites. In certain embodiments, the loxP sites have the nucleotide sequence of

[0109] 5’-ATAACTTCGTATANNNTANNNTATACGAAGTTAT (SEQ ID NO: 1) wherein N is any nucleotide.

[0110] In certain embodiments, a first pair of loxP sites have the nucleic acid sequence of 5 ’ -ATAACTTCGTATAGCATAC ATTATACGAAGTTATACTAGTGCTATCGATGTTGA GCTCAGGTACCTATAACTTCGTATAGGATACTTTATACGAAGTTAT (SEQ ID NO: 2).

[0111] In certain embodiments, a second pair of loxP sites have the nucleic acid sequence of 5 ’ -ATAACTTCGTATAATGTATGCTATACGAAGTTATC AAGCTTGCGTACGCGTGA CCGGTACTCGAGGATAACTTCGTATAAAGTATCCTATACGAAGTTAT (SEQ ID NO: 3).In certain embodiments, the viral vector is a recombinant Murine Stem Cell Virus (MSCV) vector.

[0112] In certain embodiments, this disclosure relates to a recombinant vector disclosed herein wherein the segment encoding methioninase comprises an N-terminal segment encoding a secretion sequence. In certain embodiments, the secretion sequence is an IL2 secretion sequence having the amino acid sequence of MYSMQLASCVTLTLVLLVNS (SEQ ID NO: 5) or variant thereof.

[0113] In certain embodiments, this disclosure relates to a recombinant vector disclosed herein wherein the encoded methioninase has the amino acid sequence of MRDSHNNTGFSTRAIHHGYDPLSHGGALVPPVYQTATYAFPTVEYGAACFAGEE AGHFYSRISNPTLALLEQRMASLEGGEAGLALASGMGAITSTIWTLLRPGDELIVGRTLY GCTFAFLHHGIGEFGVKIHHVDLNDAKALKAAINSKTRMIYFETPANPNMQLVDIAAVVE AVRGRDVLVVVDNTYCTPYLQRPLELGADLVVHSATKYLSGHGDITAGLVVGRKALVD RIRLEGLKDMTGAVLSPHDASLLMRGIKTLALRMDRHCANALQVAQFLARQPQVELIHY PGLPSFAQYELAQRQMRLPGGMIAFELKGGIDAGRRFMNALQLFARAVSLGDAESLAQH PASMTHS S YTPQERAHHGISEGLVRL S VGLED VEDLL AD VEQ ALQACKYP YD VPD YAEQ KLISEEDL (SEQ ID NO: 6) or variant thereof.

[0114] In certain embodiments, this disclosure relates to a recombinant vector disclosed herein wherein the nucleic acid sequence encoding the methioninase has the nucleotide sequence of: ATGTACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGTCAACA GCATGAGGGACTCACACAACAACACTGGCTTCTCTACCAGGGCCATCCATCATGGCT ACGATCCCCTGTCACATGGCGGAGCACTCGTGCCCCCTGTGTACCAGACCGCCACCT ACGCCTTCCCTACCGTGGAATATGGCGCCGCTTGCTTTGCCGGAGAGGAGGCCGGAC ACTTCTACAGCAGGATCTCTAATCCTACCCTGGCCCTGCTGGAGCAGCGGATGGCTTC TCTGGAGGGAGGCGAGGCTGGCCTGGCCCTGGCCTCTGGCATGGGCGCTATTACATC CACTATTTGGACTCTGCTGCGGCCTGGCGACGAGCTGATCGTGGGCAGGACCCTGTAT GGCTGCACTTTCGCTTTTCTGCACCACGGAATCGGCGAGTTTGGCGTGAAGATCCAC CACGTCGACCTCAATGATGCCAAAGCCCTGAAGGCTGCTATCAATTCTAAAACCAGA ATGATCTACTTCGAGACACCTGCTAATCCTAACATGCAGCTGGTGGATATCGCCGCCG TGGTGGAGGCCGTGAGAGGACGGGATGTGCTGGTCGTTGTGGACAATACATATTGCA CACCTTATCTGCAGCGGCCCCTGGAACTGGGCGCCGATCTGGTGGTGCACAGCGCAACCAAGTACCTGTCTGGGCATGGAGATATCACTGCCGGCCTGGTGGTGGGAAGAAAAG CTCTGGTTGACAGAATCCGTCTGGAGGGCCTGAAGGACATGACAGGAGCCGTGCTGA GTCCCCACGACGCCAGCCTGCTGATGAGGGGCATCAAGACCCTGGCTCTGAGAATGG ACAGGCACTGCGCAAATGCCCTCCAGGTCGCCCAGTTTCTGGCACGCCAGCCACAGG TGGAGCTGATCCACTACCCTGGCCTGCCATCCTTCGCCCAGTACGAGCTGGCTCAGA GACAGATGAGACTGCCTGGCGGTATGATTGCCTTTGAACTCAAGGGGGGCATCGACG CTGGAAGAAGATTCATGAACGCCCTGCAGCTGTTTGCCCGCGCTGTGTCACTGGGAG ACGCTGAGAGCCTGGCCCAGCACCCTGCCAGCATGACCCACTCCTCCTATACCCCTC AGGAGAGGGCTCACCACGGCATCAGCGAGGGCCTGGTGCGTCTGTCTGTGGGACTG GAGGATGTGGAGGACCTGCTGGCCGATGTGGAGCAGGCCCTGCAGGCTTGCAAGTA CCCATACGATGTTCCAGATTACGCTGAACAAAAACTCATCTCAGAAGAGGATCTGTGA

[0115] (SEQ ID NO: 7) or variants or degenerate codons encoding amino acids of a functional methioninase variant encoded therein.

[0116] In certain embodiments, this disclosure relates to a recombinant vector disclosed herein wherein the nucleic acid sequence encoding the methioninase and the pairs of loxP sites has the sequence of:

[0117] ATAACTTCGTATAGCATACATTATACGAAGTTATACTAGTGCTATCGATGTTGAGCTC AGGTACCTATAACTTCGTATAGGATACTTTATACGAAGTTATTCTAGAGCCACCATG TACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGTCAACAGCA TGAGGGACTCACACAACAACACTGGCTTCTCTACCAGGGCCATCCATCATGGCTACG ATCCCCTGTCACATGGCGGAGCACTCGTGCCCCCTGTGTACCAGACCGCCACCTACG CCTTCCCTACCGTGGAATATGGCGCCGCTTGCTTTGCCGGAGAGGAGGCCGGACACT TCTACAGCAGGATCTCTAATCCTACCCTGGCCCTGCTGGAGCAGCGGATGGCTTCTCT GGAGGGAGGCGAGGCTGGCCTGGCCCTGGCCTCTGGCATGGGCGCTATTACATCCAC TATTTGGACTCTGCTGCGGCCTGGCGACGAGCTGATCGTGGGCAGGACCCTGTATGG CTGCACTTTCGCTTTTCTGCACCACGGAATCGGCGAGTTTGGCGTGAAGATCCACCA CGTCGACCTCAATGATGCCAAAGCCCTGAAGGCTGCTATCAATTCTAAAACCAGAATG ATCTACTTCGAGACACCTGCTAATCCTAACATGCAGCTGGTGGATATCGCCGCCGTGG TGGAGGCCGTGAGAGGACGGGATGTGCTGGTCGTTGTGGACAATACATATTGCACAC CTTATCTGCAGCGGCCCCTGGAACTGGGCGCCGATCTGGTGGTGCACAGCGCAACCA AGTACCTGTCTGGGCATGGAGATATCACTGCCGGCCTGGTGGTGGGAAGAAAAGCTCTGGTTGACAGAATCCGTCTGGAGGGCCTGAAGGACATGACAGGAGCCGTGCTGAGT CCCCACGACGCCAGCCTGCTGATGAGGGGCATCAAGACCCTGGCTCTGAGAATGGAC AGGCACTGCGCAAATGCCCTCCAGGTCGCCCAGTTTCTGGCACGCCAGCCACAGGTG GAGCTGATCCACTACCCTGGCCTGCCATCCTTCGCCCAGTACGAGCTGGCTCAGAGA CAGATGAGACTGCCTGGCGGTATGATTGCCTTTGAACTCAAGGGGGGCATCGACGCT GGAAGAAGATTCATGAACGCCCTGCAGCTGTTTGCCCGCGCTGTGTCACTGGGAGAC GCTGAGAGCCTGGCCCAGCACCCTGCCAGCATGACCCACTCCTCCTATACCCCTCAG GAGAGGGCTCACCACGGCATCAGCGAGGGCCTGGTGCGTCTGTCTGTGGGACTGGA GGATGTGGAGGACCTGCTGGCCGATGTGGAGCAGGCCCTGCAGGCTTGCAAGTACCC ATACGATGTTCCAGATTACGCTGAACAAAAACTCATCTCAGAAGAGGATCTGTGACCT AGGATAACTTCGTATAATGTATGCTATACGAAGTTATCAAGCTTGCGTACGCGTGAC CGGTACTCGAGGATAACTTCGTATAAAGTATCCTATACGAAGTTAT (SEQ ID NO: 8) or variants or degenerate codons encoding amino acids of a functional methioninase encoded therein.

[0118] In certain constructs, expression is regulated by Cre or Tetracycline / Dox to prevent harming normal cells. The design targets tumor cells and nearby cells. It is controlled by a Cre system wherein the DNA coding sequence of bacterial methioninase was optimized preserving the encoded amino acid sequence. The bacterial methioninase gene was placed into a Cre-conditional MSCV virus. A secretion signal sequences was added to the construct to promote extracellular secretion of the enzyme. Cre expression (Cre-lox system) provides a safety switch to prevent widespread methioninase expression. This feature is advantageous because normal cells in the brain need methionine. Data indicates that Cre induction causes methioninase expression (intracellular) and secretion (extracellular). Data also indicates that Cre induction of methioninase blocks the growth of human cell line HEK293.

[0119] Another contemplated design targets macrophages that home to tumors, controlled by a Tet (tetracycline) system. Tetracycline molecules comprise a linear fused tetracyclic nucleus (carbocyclic rings typically designated A, B, C and D) to which a variety of functional groups are attached. See doxycycline having he chemical name (4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-l,ll-dioxo-l,4,4a,5,5a,6,ll,12a-octahydrotetracene-2-carboxamide). The tetracycline-controlled Tet-Off and Tet-On gene expression systems are used to regulate the activity of genes in eukaryotic cells which provide doxycycline-inducible gene expression, i.e. by the administration of tetracycline / doxycycline based agents.TRE is used as a Tet responsive element for inhibiting inappropriate expression of methioninase. ATG is the start site of translation for the enzyme. IL2 is the secretion signal indicating to the cells that the enzyme is to be secreted. B7H3 binding domain is an antigen binding domain specific to the cancer cells such that one half of a recombinant protein will bind the cancer cell surface and the other half will degrade methionine extracellularly. The designs can be transduced into macrophages ex vivo then injected in vivo.

[0120] In certain embodiments, this disclosure relates to a recombinant vector or recombinant vector as disclosed herein comprising a segment encoding Tet-on METase, i.e., methioninase in operable combination with a Tet / Dox promoter and optionally two flanking LoxP sites. In certain embodiments, this disclosure contemplates expression of methioninase using a tetracycline (Tet)-or doxycycline (Dox; a tetracycline derivativej-inducible Cre system. Dox can be used as a substituted for tetracycline for controlling the Tet receptor (TetR). This system is available in two modes, Tet-on and Tet-off, which permit Dox-dependent gene activation or inactivation. The Tet systems consist of three elements, reverse tetracycline controlled transactivator (rtTA), tetracycline-controlled transactivator (tTA) and tetracycline responsive element (TRE), also referred to as a tetracycline operon (TetO), which regulates a cre gene expression. When the rtTA binds to Tet / Dox, it can bind to the tetO7 (7 repeats of tetO) sequences and activates Cre gene expression (“Tet-on”), or on the other hand, if tTA binds to tetO7 sequences as normally, and when it is coupled with Dox, it can no longer bind to the tetO7 sequences and inactivates Cre expression (“Tet-off’). In Tet system, Tet / doxycycline is used / administered.

[0121] Intratumoral injection of drugs into the brain evades the blood-brain-barrier and reduces systemic side effects. Engineering macrophages to deliver drugs to tumor sites in a targeted manner is a promising strategy for cancer treatment. This approach can enhance the effectiveness of current therapies and reduce side effects.

[0122] Disclosed herein is a recombinant virus that induces expression of the bacterial enzyme methioninase in mammalian cells, for the purpose of treating brain tumors. Methionine is an enzyme that degrades the amino acid methionine, and this enzyme is not expressed in any mammalian cells. The pediatric brain tumor called medulloblastoma depends on robust supply of methionine, and that dietary methionine deprivation impairs tumor growth. To induce stronger methionine deprivation than can be achieved through diet, and to limit methionine deprivation to the tumor microenvironment, recombinant retroviral constructs were designed that can transducecells to express and secrete bacterial methioninase. The DNA sequence of pseudomonas methioninase was altered by optimizing codon utilization while preserving the encoded amino acid sequence. Secretion signal sequences 5' were added to the methioninase sequence, and a Myc tag was added a to the 3' end to facilitate detection of the expressed protein. The coding region was then placed into Cre-Conditional MSCV and lentiviral plasmids, using the requirement for Cre expression as a vital safety switch to prevent more widespread methioninase expression than intended. These plasmids were used to generate MSCV and lentivirus particles that transduce mammalian cells. The methioninase inducing viruses were tested. Cre-conditional expression and secretion of methioninase protein was verified. These viruses stop the growth of cancer cells.

Claims

CLAIMS1. A recombinant vector comprising a segment encoding methioninase and two pairs of loxP sites.

2. The recombinant vector of claim 1, wherein the loxP sites have the nucleotide sequence of 5 -ATAACTTCGTATANNNTANNNTATACGAAGTTAT (SEQ ID NO: 1) wherein N is any nucleotide.

3. The recombinant vector of claim 1, wherein first pair of loxP sites have the nucleic acid sequence of5 ’ -ATAACTTCGTATAGCATAC ATTATACGAAGTTATACTAGTGCTATCGATGTTGAG CTCAGGTACCTATAACTTCGTATAGGATACTTTATACGAAGTTAT (SEQ ID NO: 2).

4. The recombinant vector of claim 1, wherein second pair of loxP sites have the nucleic acid sequence of 5 -ATAACTTCGTATAATGTATGCTATACGAAGTTATCAAGCTTGCGTACG CGTGACCGGTACTCGAGGATAACTTCGTATAAAGTATCCTATACGAAGTTAT (SEQ ID NO: 3).

5. The recombinant vector of any of claims 1-4 wherein the segment encoding methioninase comprises an N-terminal segment encoding a secretion sequence.

6. The recombinant vector of claim 5, wherein the secretion sequence is an IL2 secretion sequence having the amino acid sequence of MYSMQLASCVTLTLVLLVNS (SEQ ID NO: 5) or variant thereof.

7. The recombinant vector of any of claims 1-6, wherein the methioninase has the amino acid sequence of MRDSHNNTGFSTRAIHHGYDPLSHGGALVPPVYQTATYAFPTVEYGAACFAGEEAGHFY SRISNPTLALLEQRMASLEGGEAGLALASGMGAITSTIWTLLRPGDELIVGRTLYGCTFAF LHHG1GEFGVK1HHVDLNDAKALKAA1NSKTRMIYFETPANPNMQLVDIAAVVEAVR.GR D VLVVVDNT YCTP YLQ R PLF.LG A DLV VH S ATK YL SGHGDITAGLVVGRK A LVDR I R LEGLKDMTGAVLSPHDASLLMRGIKTLALRMDRHCANALQVAQFLARQPQVELIHYPGLPSF AQYELAQRQMRLPGGMIAFELKGGIDAGRRFMNALQLFARAVSLGDAESLAQHPASMT HSSYTPQERAHHGISEGLVRLSVGLEDVEDLLADVEQALQACKYPYDVPDYAEQKLISE EDL (SEQ ID NO: 6) or variant thereof.

8. The recombinant vector of any of claims 1-7 comprising the nucleic acid sequence of ATGTACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGTC AACAGCATGAGGGACTCACACAACAACACTGGCTTCTCTACCAGGGCCATCCATCAT GGCTACGATCCCCTGTCACATGGCGGAGCACTCGTGCCCCCTGTGTACCAGACCGCC ACCTACGCCTTCCCTACCGTGGAATATGGCGCCGCTTGCTTTGCCGGAGAGGAGGCC GGACACTTCTACAGCAGGATCTCTAATCCTACCCTGGCCCTGCTGGAGCAGCGGATGG CTTCTCTGGAGGGAGGCGAGGCTGGCCTGGCCCTGGCCTCTGGCATGGGCGCTATTA CATCCACTATTTGGACTCTGCTGCGGCCTGGCGACGAGCTGATCGTGGGCAGGACCC TGTATGGCTGCACTTTCGCTTTTCTGCACCACGGAATCGGCGAGTTTGGCGTGAAGAT CCACCACGTCGACCTCAATGATGCCAAAGCCCTGAAGGCTGCTATCAATTCTAAAACC AGAATGATCTACTTCGAGACACCTGCTAATCCTAACATGCAGCTGGTGGATATCGCCG CCGTGGTGGAGGCCGTGAGAGGACGGGATGTGCTGGTCGTTGTGGACAATACATATT GCACACCTTATCTGCAGCGGCCCCTGGAACTGGGCGCCGATCTGGTGGTGCACAGCG CAACCAAGTACCTGTCTGGGCATGGAGATATCACTGCCGGCCTGGTGGTGGGAAGAA AAGCTCTGGTTGACAGAATCCGTCTGGAGGGCCTGAAGGACATGACAGGAGCCGTG CTGAGTCCCCACGACGCCAGCCTGCTGATGAGGGGCATCAAGACCCTGGCTCTGAGA ATGGACAGGCACTGCGCAAATGCCCTCCAGGTCGCCCAGTTTCTGGCACGCCAGCCA CAGGTGGAGCTGATCCACTACCCTGGCCTGCCATCCTTCGCCCAGTACGAGCTGGCTC AGAGACAGATGAGACTGCCTGGCGGTATGATTGCCTTTGAACTCAAGGGGGGCATCG ACGCTGGAAGAAGATTCATGAACGCCCTGCAGCTGTTTGCCCGCGCTGTGTCACTGG GAGACGCTGAGAGCCTGGCCCAGCACCCTGCCAGCATGACCCACTCCTCCTATACCC CTCAGGAGAGGGCTCACCACGGCATCAGCGAGGGCCTGGTGCGTCTGTCTGTGGGA CTGGAGGATGTGGAGGACCTGCTGGCCGATGTGGAGCAGGCCCTGCAGGCTTGCAA GTACCCATACGATGTTCCAGATTACGCTGAACAAAAACTCATCTCAGAAGAGGATCTG TGA (SEQ ID NO: 7) variants or variant codons encoding amino acids of a functional methioninase encoded therein.

9. The recombinant vector of any of claims 1-7 comprising the nucleic acid sequence of ATAACTTCGTATAGCATACATTATACGAAGTTATACTAGTGCTATCGATGTTGAGCTC AGGTACCTATAACTTCGTATAGGATACTTTATACGAAGTTATTCTAGAGCCACCATG TACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGTCAACAGCA TGAGGGACTCACACAACAACACTGGCTTCTCTACCAGGGCCATCCATCATGGCTACG ATCCCCTGTCACATGGCGGAGCACTCGTGCCCCCTGTGTACCAGACCGCCACCTACG CCTTCCCTACCGTGGAATATGGCGCCGCTTGCTTTGCCGGAGAGGAGGCCGGACACT TCTACAGCAGGATCTCTAATCCTACCCTGGCCCTGCTGGAGCAGCGGATGGCTTCTCT GGAGGGAGGCGAGGCTGGCCTGGCCCTGGCCTCTGGCATGGGCGCTATTACATCCAC TATTTGGACTCTGCTGCGGCCTGGCGACGAGCTGATCGTGGGCAGGACCCTGTATGG CTGCACTTTCGCTTTTCTGCACCACGGAATCGGCGAGTTTGGCGTGAAGATCCACCA CGTCGACCTCAATGATGCCAAAGCCCTGAAGGCTGCTATCAATTCTAAAACCAGAATG ATCTACTTCGAGACACCTGCTAATCCTAACATGCAGCTGGTGGATATCGCCGCCGTGG TGGAGGCCGTGAGAGGACGGGATGTGCTGGTCGTTGTGGACAATACATATTGCACAC CTTATCTGCAGCGGCCCCTGGAACTGGGCGCCGATCTGGTGGTGCACAGCGCAACCA AGTACCTGTCTGGGCATGGAGATATCACTGCCGGCCTGGTGGTGGGAAGAAAAGCTC TGGTTGACAGAATCCGTCTGGAGGGCCTGAAGGACATGACAGGAGCCGTGCTGAGT CCCCACGACGCCAGCCTGCTGATGAGGGGCATCAAGACCCTGGCTCTGAGAATGGAC AGGCACTGCGCAAATGCCCTCCAGGTCGCCCAGTTTCTGGCACGCCAGCCACAGGTG GAGCTGATCCACTACCCTGGCCTGCCATCCTTCGCCCAGTACGAGCTGGCTCAGAGA CAGATGAGACTGCCTGGCGGTATGATTGCCTTTGAACTCAAGGGGGGCATCGACGCT GGAAGAAGATTCATGAACGCCCTGCAGCTGTTTGCCCGCGCTGTGTCACTGGGAGAC GCTGAGAGCCTGGCCCAGCACCCTGCCAGCATGACCCACTCCTCCTATACCCCTCAG GAGAGGGCTCACCACGGCATCAGCGAGGGCCTGGTGCGTCTGTCTGTGGGACTGGA GGATGTGGAGGACCTGCTGGCCGATGTGGAGCAGGCCCTGCAGGCTTGCAAGTACCC ATACGATGTTCCAGATTACGCTGAACAAAAACTCATCTCAGAAGAGGATCTGTGACCT AGGATAACTTCGTATAATGTATGCTATACGAAGTTATCAAGCTTGCGTACGCGTGAC CGGTACTCGAGGATAACTTCGTATAAAGTATCCTATACGAAGTTAT (SEQ ID NO: 8).

10. The recombinant vector of any of claims 1 -7 comprising the nucleic acid sequence of (SEQ ID NO: 4) variants or variant codons encoding amino acids of proteins encoded therein.

11. A recombinant vector as in any of claims 1-10 further comprising a TRE promoter and a start codon encoding a secretion sequence and a methioninase and a human influenza hemagglutinin (HA) sequence.

12. The recombinant vector of claim 11 further encoding a domain that specially binds a tumor specific antigen.

13. The recombinant vector of claim 12 wherein the domain that specifically binds a tumor specific antigen is B7-H3 binding domain.

14. The recombinant vector of claim 13, wherein the B7-H3 binding domain is linked to the N-terminal of methioninase and optionally HA is linked to the C-terminal of methioninase providing a fusion protein.

15. A method of treating cancer comprising administering an effective amount of Cre enzyme or vector encoding a Cre enzyme and a recombinant Murine Stem Cell Virus (MSCV) vector of any of claims 1-14 to a subject in need thereof.

16. The method of claim 15, wherein the cancer is medulloblastoma.

17. A method of treating cancer comprising transducing cells ex vivo with Cre enzyme or vector encoding a Cre enzyme and a recombinant Murine Stem Cell Virus (MSCV) vector of any of claims 1-14 administering an effective amount of the transduced cells to a subject in need thereof.

18. The method of claim 17 wherein the cells are macrophages.

19. The method of claim 17, wherein the cancer is a medulloblastoma.

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